Blood serum reference standard for multi-automated analytical procedures



United States Patent 3,466,249 BLOOD SERUM REFERENCE STANDARD FOR MULTI-AUTOMATED ANALYTICAL PROCEDURES Roy A. Anderson, North Hollywood, Calif., assignor to Baxter Laboratories, Inc., Morton Grove, 11]., a corporation of Delaware No Drawing. Filed Feb. 13, 1967, Ser. No. 615,339 Int. Cl. C09k 3/00 U.S. Cl. 252-408 4 Claims ABSTRACT OF THE DISCLOSURE A stable blood serum reference standard having a standard carbon dioxide content for use in multi-automated analytical procedures in which a freeze-dried blood serum component of the reference standard is reconstituted with an aqueous ammonium bicarbonate component of the reference standard.

This invention relates to a laboratory standard material and, more particularly, to a stable blood serum reference standard and to a method of preparation and use thereof.

The rapid quantitation or estimation of various blood serum components has become a valuble adjunct in the clinical diagnosis of disease. In recent years a number of elaborate electronic devices have been developed for making rapid and precise analytical determinations on blood serum and other biochemical samples. An automatic analyzer known as the Robot Chemist made by Warner-Chilcott Laboratories and various instruments known by the name AutoAnalyzer made by Technicon Instruments are examples of such electronic devices capable of making rapid and precise analytical determinations. Some of the various biochemical determinations which the above and similar electronic devices are presently adaptable to are, for example, albumin, acid phosphatase, alkaline, phosphatase, bilirubin, calcium, carbon dioxide, chloride, cholesterol, glucose, hemoglobin, LDH, potassium, sodium, total protein, transaminase, urea nitrogen and uric acid. One particular instrument available on the market (known as the 12 Channel AutoAnalyzer) is capable of simultaneously making twelve different analyses on a single sample.

In the performance of the analytical tests made by the above and similar electronic devices on blood serum and other biological samples, it is necessary to use laboratory standard materials or so-called reference standards for purposes of calibration and control of the instrument. Accurate results in the use of these instruments, particularly in the case of multi-automated procedures, are dependent upon rigid and constant standardization of the biochemical determinations.

Since blood is a complex material which contains numerous substances, multi-automated procedures lend themselves ideally to the analysis of blood. Of the many functions of blood, an important one is to carry raw materials to the tissues and remove the waste products therefrom. One of the most essential of these raw materials is oxygen, and an important waste product is carbon dioxide which the hemoglobin of the red cells carries away from the tissues to which it has delivered oxygen. Also, the carbon dioxide in normal blood is maintained in equilibrium with H CO and HCO to comprise an important buffering system for controlling the pH of the blood. Consequently, determination of the carbon dioxide content of the blood serum is of great importance in many clinical procedures. A multi-automated procedure which includes an arcuate determination of the carbon ice dioxide content of blood would, therefore, be very useful in the clinical laboratory.

Accordingly, it is an object of this invention to provide a stable blood serum reference standard which includes a standard carbon dioxide content.

It is another object of this invention to provide a stable blood serum reference standard which includes a standand carbon dioxide content and which can be used for calibration and control of multi-automated analytical procedures.

As used herein, the term carbon dioxide content refers to the total equivalent amount of carbon dioxide in the form of CO H CO and HCO As used herein, the term multi-automated procedure refers to an automated chemical analytical procedure in which two or more analyses are made simultaneously on a single sample.

In brief, the blood serum reference standard of the present invention comprises a freeze-dried serum prepared from human blood and a separate aqueous solution of ammonium bicarbonate for use in reconstitution of the freeze-dried serum.

The freeze-dried serum used in the practice of this invention is stable for at least two years at temperatures ranging from about 2 to about 10 C. In the method of this invention, the freeze-dried serum is reconstituted with sufficient aqueous ammonium bicarbonate solution (a) to provide from about 12 to about 35 meq./liter of carbon dioxide content in the reconstituted serum or (b) to increase the carbon dioxide content of the reconstituted serum to any predetermined level, depending upon the particular analytical procedure concerned. This reconstituted serum is stable for at least three days at temperatures ranging from about 2 to about 10 C., and is directly used for the calibration and control of the instruments used in the multi-automated analytical procedures.

The freeze-dried serum used in the practice of the present invention can be prepared by any conventional method of freeze-drying whereby the serum is dried from the frozen state under a high vacuum and ice or other frozen solvent rapidly sublimes to yield a porous solid.

The use of the separate aqueous solution of the ammonium bicarbonate for reconstitution of the freeze-dried serum has been found necessary according to the present invention to provide a stable blood serum reference standard. It has been found that in the freeze-drying of the blood serum, a substantial amount of the carbon dioxide content of the serum is lost in the form of gaseous CO And in order to preserve enzymes in the serum, acid is added to provide the optimum pH at which the enzymes remain active. This acidification causes even further losses in the carbon dioxide content of the serum.

The ammonium bicarbonate reconstituting solution is uniquely suited as a component of the blood serum reference standard of this invention in that:

(1) It is stable in solution when hermetically sealed and sterilized.

(2) It has a composition that does not cause interference with measurement of any of the constituents of blood ordinarily tested in multi-automated analytical procedures.

(3) It has a composition that does not undesirably contribute constituents which would throw otf any of the ordinary target values for the blood serum reference standard, i.e., values substantially the same as in normal human blood serum or values ideally suited to multi-automated analytical procedures.

(4) It has a pH in such a range that the final pH (e.g., pH 7510.5) of the reconstituted serum is within an acceptable range for the test systems as well as for stability of the components, particularly the enzymes.

(5) It contributes to the carbonate-bicarbonate system when added to the serum to produce a carbon dioxide content that is suitable for standardizing and controlling the test.

(6) It has no color and can be filtered to produce a water-clear solution.

The following example will further illustrate the present invention although the invention is not limited to this specific example which is provided by Way of illustration and not limitation.

EXAMPLE A solution of ammonium bicarbonate was prepared by dissolving 426.6 g. of ammonium bicarbonate (A.R. grade) in 180 liters of distilled water. The solution was clarified by passage through Millipore membranes (1.2 and 0.45 micron), filled into 50 ml. bottles, stoppered, hermetically sealed and then sterilized by autoclaving at 255 F. for 30 minutes.

Freeze-dried human blood serum representing an original volume of 50 m1. of liquid serum was reconstituted to its original volume by the addition of 50 ml. of the above ammonium bicarbonate solution. The mixture was slightly agitated by rotation of the bottle to completely wet the serum with the aqueous solution and then placed in a refrigerator until the serum was completely dissolved. A sample of the reconstituted serum had the following analysis:

Constituent: Mean value Albumin, g./100 ml. 3.9 Alkaline phosphatase, units 18 1 Bilirubin, mg./l00 ml. 1.8 Calcium, mg./ 100 ml. 9.0 Chloride, meq./l. 101 Carbon dioxide content, meq./l. 25 1 Glucose, mg./100 ml. 230 Potassium, rneq./l. 5.0 Sodium, meq./l. 140 Total protein, g./100 ml. 6.8 1 Transaminase (GOT), units 100 Urea nitrogen, mg./l00 ml. 68

These values are in an elevated range. The liquid serum was preadjusted (prior to freeze-drying) by the addition of pure compounds and other substances to give values in certain ideal ranges for standardizing nlulti-channel automated instruments such as the "12 Channel AutoAnalyzer.

The above reconstituted serum can be used for the standardization and control of multi-automated analytical procedures for blood serum such as with the 12 Channel AutoAnalyzer made by Technicon Instruments.

As will be readily apparent to those skilled in the art,

other examples of the herein-defined invention can be devised after reading the foregoing specification and the claims appended hereto by various modifications, variations and adaptations without departing from the spirit and scope of the invention. All such modifications, variations and adaptations are included within the scope of the invention as defined in the appended claims.

What is claimed is:

1. A blood serum reference standard comprising a first container of freeze-dried blood serum and a second container of aqueous ammonium bicarbonate, the concentration of said aqueous ammonium bicarbonate being such that when the serum is reconstituted with the aqueous ammoniurn bicarbonate the carbon dioxide content of the reconstituted serum is from about 12 to about meq./ liter.

2. The method of providing an accurate blood serum reference standard including a standard carbon dioxide content for use in multi-automated analytical procedures comprising reconstituting freeze-dried blood serum with an aqueous solution of ammonium bicarbonate such that the carbon dioxide content of the reconstituted serum is increased to a predetermined level.

3. The method of providing an accurate blood serum 0 reference standard including a standard carbon dioxide UNITED STATES PATENTS 2,225,744 12/1940 Flosdorf 252-408 2,770,602 11/1956 Weichselbaum 252-408 3,000,836 9/1961 Ginsburg 252408 3,147,081 9/1964 Stevenson et a1.

MORRIS O. WOLK, Primary Examiner R. E. SERWIN, Assistant Examiner US. Cl. X.R. 

